The invention relates to isoelectric point markers and a gel isoelectric separation method by employing isoelectric point markers. More particularly this invention relates to a gel isoelectric separation method for measuring the isoelectric point of proteins by employing a combination of colored proteins of which the isoelectric points are known.
Recently, in the fields of biochemistry and clinical diagnosis a gel isoelectric separation method (also named a gel isoelectric focusing method) has been taken note of as a method for separating, purifying and analyzing proteins. This method is considered to be a simplified method of the density gradient isoelectric separation method, which was developed by Svensson et al. When a mixture of special ampholytes having different isoelectric points at different pH values is in a gel such as acrylamide and agar-agar, and an electric current is applied between both ends of the gel, a pH gradient derived from the ampholytes is developed between the electrodes placed at both ends. This method is based on the principle that under the influence of an electric field, proteins are focused at their own isoelectric points in the pH gradient. (C. W. Wringley : Method in Enzymology, Vol. 22, page 559-564, Academic Press) According to this invention, it is possible not only to separate and purify proteins by utilizing the differences among the isoelectric points of the proteins, but also to determine isoelectric points of proteins by measuring the pH gradient in a gel. Therefore, this method is very useful as a method of analyzing proteins.
There, however, are some disadvantages in this method. One disadvantage is that a complicated operation is required in determining the pH gradient formed in a gel as described below. As soon as electrophoresis ends, the gel is removed rapidly and sliced as thinly as possible at equal intervals. Thereafter ampholytes enclosed in the resulting sliced gel intervals are extracted with pure and decarbonated water and then each pH value of the extract is measured. In this pH determination, it takes time to extract ampholytes and it is difficult to slice the gel accurately at equal intervals without failure. Furthermore, the resulting pH values vary with the conditions of extraction. Therefore this method is not applicable as a practical method. Hence, there are reported some modified methods, such as a method wherein amphoteric dyes are used together with samples as pH indicators in a gel isoelectric separation method (A. Conway-Jacobs and L. M. Lewin : Analytical Biochemistry, Vol. 43, page 394-400(1971)), and a method wherein phenanthroline-iron complexes are used for the same purpose. These methods, however, that the disadvantages that the compounds used are diffused rapidly because they are low molecular compounds and they do not focus sharply because the ionization constants of the ionized groups at opposite ends of the compounds are greatly different from each other.